Methods and materials for treating renal cell carcinoma

ABSTRACT

This document provides methods and materials related to treating renal cell carcinoma. For example, methods and materials for assessing a cancer patient (e.g., a renal cell carcinoma patient) for tumor or peritumoral tissue containing CD14 +  cells and proceeding with a cancer treatment option (e.g., a renal cell carcinoma treatment option) based on the presence, absence, or level of CD14 +  cells present within the tumor or peritumoral tissue are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/643,155, filed Jul. 6, 2017, which is a continuation of U.S.application Ser. No. 14/994,980, filed Jan. 13, 2016 (now U.S. Pat. No.9,733,248), which is a continuation of U.S. application Ser. No.13/509,089, filed May 10, 2012 (now U.S. Pat. No. 9,266,957), which is aNational Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2010/055856, having an International filing dateof Nov. 8, 2010, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/259,879, filed Nov. 10, 2009. The disclosures ofthe prior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in treatingrenal cell carcinoma. For example, this document provides methods andmaterials for assessing a renal cell carcinoma patient for tumor orperitumoral tissue containing CD14⁺ cells and proceeding with a renalcell carcinoma treatment option based on the presence, absence, or levelof CD14⁺ cells present within the tumor or peritumoral tissue.

2. Background Information

The incidence and deaths caused by renal cell carcinoma are increasingin the United States. Of particular note, incidence and mortality ratesfor renal cell carcinoma have risen among both genders, and these trendsare not explained by the increased use of abdominal imaging (Chow etal., JAMA, 281:1628-31 (1999)). Indeed, mortality from renal cellcarcinoma has increased over 30% since 1950. One treatment for renalcell carcinoma is surgical resection. The majority of patients withrenal cell carcinoma confined to the kidney can be cured by surgery;however, about 30 percent of patients can develop metastases and requireadditional treatments.

Renal cell carcinoma encompasses a group of at least five subtypes withunique morphologic, genetic, and behavioral characteristics (Cheville etal., Am. J. Surg. Pathol., 27:612-24 (2003)). Cancer-specific survivalis dependent on subtype, and over 80 percent of renal cell carcinomasand the vast majority of renal cell carcinoma-related deaths are due toclear cell renal cell carcinoma. To date, tumor stage and grade are theprimary prognostic indicators for patients with clear cell renal cellcarcinoma treated by nephrectomy (Gettman et al., Cancer, 91:354-61(2001)). There is, however, variability in patient outcome that cannotbe explained by the combination of stage and grade.

SUMMARY

This document relates to methods and materials involved in treatingrenal cell carcinoma. For example, this document provides methods andmaterials for assessing a renal cell carcinoma patient for tumor orperitumoral tissue containing CD14⁺ cells and proceeding with a renalcell carcinoma treatment option based on the presence, absence, or levelof CD14⁺ cells present within the tumor or peritumoral tissue.

As described herein, the presence of a moderate to marked level of CD14⁺cells within tumor or peritumoral tissue of a renal cell carcinomapatient can indicate that it is unlikely that the renal cell carcinomapatient will respond positively to a standard renal cell carcinomatreatment that is appropriate for renal cell carcinoma patients lackinga moderate to marked level of CD14⁺ cells within their tumor andperitumoral tissue. A standard renal cell carcinoma treatment that isappropriate for renal cell carcinoma patients lacking a moderate tomarked level of CD14⁺ cells within their tumor and peritumoral tissuecan be referred to as a type A treatment. The presence of a moderate tomarked level of CD14⁺ cells within tumor or peritumoral tissue of arenal cell carcinoma patient also can indicate that it is likely thatthe renal cell carcinoma patient will respond positively to a renal cellcarcinoma treatment that is appropriate for renal cell carcinomapatients having a moderate to marked level of CD14⁺ cells within theirtumor and peritumoral tissue. A renal cell carcinoma treatment that isappropriate for renal cell carcinoma patients having a moderate tomarked level of CD14⁺ cells within their tumor and peritumoral tissuecan be referred to as a type B treatment. As also described herein, theabsence of a moderate to marked level of CD14⁺ cells within tumor orperitumoral tissue of a renal cell carcinoma patient can indicate thatit is likely that the renal cell carcinoma patient will respond to atype A treatment and/or that it is not necessary to proceed with a typeB treatment.

The methods and materials provided herein can allow clinicians todetermine proper treatment options for cancer patients (e.g., renal cellcarcinoma patients). For example, the methods and materials providedherein can be used to identify renal cell carcinoma patients who wouldbenefit from a type A treatment or a type B treatment. In some cases,the methods and materials provided herein can be used to identify renalcell carcinoma patients who would benefit from switching from a type Atreatment to a type B treatment. Identifying cancer patients who wouldbenefit from a particular treatment option can avoid unnecessarytreatment attempts and unnecessary patient suffering.

In general, one aspect of this document features a method for treating ahuman having renal cell carcinoma. The method comprises, or consistsessentially of, (a) determining whether or not tumor or peritumoraltissue from the human contains greater than 30 percent of CD14⁺ cells,and (b) proceeding with a type B treatment if the tissue containsgreater than 30 percent of CD14⁺ cells, and proceeding with a type A ortype B treatment if the tissue does not contain greater than 30 percentof CD14⁺ cells. The renal cell carcinoma can be clear cell renal cellcarcinoma. The step (a) can comprise determining whether or not thetumor tissue contains greater than 30 percent of CD14⁺ cells. The step(a) can comprise determining whether or not the peritumoral tissuecontains greater than 30 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 35 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 40 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 30 percent of CD14⁺ cells in a diffuse stainingpattern. The tumor or peritumoral tissue from the human can containgreater than 30 percent of CD14⁺ cells. The method can compriseproceeding with a type B treatment selected from the group consisting ofchemotherapies, biologic therapies, and cell therapies. The method cancomprise proceeding with a type B treatment selected from the groupconsisting of treatment with Sunitinib, treatment with Sorafenib,treatment with Bevacizumab, treatment with Temsirolimus, and treatmentwith Everolimus. The method can comprise initiating a type B treatmentfor the human after the step (a). The tumor or peritumoral tissue fromthe human can lack greater than 30 percent of CD14⁺ cells. The methodcan comprise performing a surgical treatment after the step (a).

In another aspect, this document features a method for treating a humanhaving renal cell carcinoma. The method comprises, or consistsessentially of, (a) determining whether or not tumor or peritumoraltissue from the human contains greater than 10 percent of CD14⁺ cells,and (b) proceeding with a type B treatment if the tissue containsgreater than 10 percent of CD14⁺ cells, and proceeding with a type A ortype B treatment if the tissue does not contain greater than 10 percentof CD14⁺ cells. The renal cell carcinoma can be clear cell renal cellcarcinoma. The step (a) can comprise determining whether or not thetumor tissue contains greater than 10 percent of CD14⁺ cells. The step(a) can comprise determining whether or not the peritumoral tissuecontains greater than 10 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 15 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 20 percent of CD14⁺ cells. The step (a) cancomprise determining whether or not the tumor or peritumoral tissuecontains greater than 10 percent of CD14⁺ cells in a diffuse stainingpattern. The tumor or peritumoral tissue from the human can containgreater than 10 percent of CD14⁺ cells. The method can compriseproceeding with a type B treatment selected from the group consisting ofchemotherapies, biologic therapies, and cell therapies. The method cancomprise proceeding with a type B treatment selected from the groupconsisting of treatment with Sunitinib, treatment with Sorafenib,treatment with Bevacizumab, treatment with Temsirolimus, and treatmentwith Everolimus. The method can comprise initiating a type B treatmentfor the human after the step (a). The tumor or peritumoral tissue fromthe human can lack greater than 10 percent of CD14⁺ cells. The methodcan comprise performing a surgical treatment after the step (a).

In another aspect, this document features a method for treating a humanhaving glioblastoma multiforme. The method comprises, or consistsessentially of, (a) determining whether or not tumor or peritumoraltissue from the human contains greater than 10 percent of CD14⁺ cells,and (b) proceeding with a conventional glioblastoma multiforme therapyif the tissue does not contain greater than 10 percent of CD14⁺ cells,and proceeding with a therapy more aggressive than the conventionalglioblastoma multiforme therapy if the tissue contains greater than 10percent of CD14⁺ cells. The conventional glioblastoma multiforme therapycan include temozolomide chemotherapy. The conventional glioblastomamultiforme therapy can include temozolomide chemotherapy after surgery.The conventional glioblastoma multiforme therapy can includetemozolomide chemotherapy after radiotherapy. The conventionalglioblastoma multiforme therapy can include temozolomide chemotherapyafter surgery and radiotherapy. The therapy more aggressive than theconventional glioblastoma multiforme therapy can be an experimentaltherapy.

In another aspect, this document features a method for identifying apotential treatment agent for reducing cancer aggressiveness. The methodcomprises, or consists essentially of, (a) administering a test agent toa mammal having cancer, wherein tumor or peritumoral tissue of thecancer contains a detectable level of CD14⁺ cells, and (b) determiningwhether or not the level of CD14⁺ cells present in the tumor orperitumoral tissue of the cancer is reduced as compared to the level ofCD14⁺ cells present in the tumor or peritumoral tissue of the cancerprior to the administering step (a), wherein a reduction in the level ofCD14⁺ cells present in the tumor or peritumoral tissue of the cancerindicates that the test agent is the potential treatment agent forreducing cancer aggressiveness. The mammal can be a human. The testagent can be a small molecule compound. The cancer can be a renal cellcarcinoma. The cancer can be a clear cell renal cell carcinoma. The step(b) can comprise determining whether or not the level of CD14+ cellspresent in a tumor tissue biopsy of the cancer obtained from the mammalafter the administering step (a) is reduced. The step (b) can comprisedetermining whether or not the level of CD14+ cells present in aperitumoral tissue biopsy of the cancer obtained from the mammal afterthe administering step (a) is reduced.

In another aspect, this document features a method for treating a humanhaving a solid tumor. The method comprises, or consists essentially of,(a) determining whether or not tumor or peritumoral tissue from thehuman contains greater than 30 percent of CD14⁺ cells, and (b)proceeding with a conventional ovarian cancer therapy if the tissue doesnot contain greater than 10 percent of CD14⁺ cells, and proceeding witha therapy more aggressive than the conventional ovarian cancer therapyif the tissue contains greater than 10 percent of CD14⁺ cells. The solidtumor can be a renal cell carcinoma.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an example of renal cell tumor tissue stainedfor CD14 expression demonstrating low or absent CD14 staining. Renalcell carcinoma tissue was obtained from a renal cell carcinoma patienthaving a low SSIGN score (e.g., SSIGN score of 2 or less) and stainedwith an anti-CD14 antibody.

FIG. 2 is a photograph an example of renal cell carcinoma tissue stainedfor CD14 expression demonstrating high CD14 staining. Renal cellcarcinoma tissue was obtained from a renal cell carcinoma patient havinga high SSIGN score (e.g., SSIGN score of 7 or more) and stained with ananti-CD14 antibody.

FIG. 3. Cancer-specific survival following surgery by maximalintratumoral and peritumoral CD14 expression for 375 ccRCC patients.Estimated cancer-specific survival rates (95% CI; number still at risk)at 5 years following surgery were 100% (100-100; 6), 92.2% (88.2-96.4;134), 64.1% (56.4-73.0; 75), and 41.8% (29.6-58.9; 17) for patients withtumors with absent, focal, moderate, and marked CD14 expression,respectively.

FIG. 4 is a graph plotting the HLA-DR mean fluorescence intensity (MFI)of peripheral blood cells that are CD14⁺ and obtained from patientshaving tumor tissue with a high intensity of CD14 staining (high), amedium intensity of CD14⁺ staining (medium), or a low/no intensity ofCD14⁺ staining (low/none).

DETAILED DESCRIPTION

This document provides methods and materials related to treating solidtumors (e.g., renal cell carcinomas and ovarian cancers). For example,this document provides methods and materials for assessing a renal cellcarcinoma patient for tumor or peritumoral tissue containing CD14⁺ cellsand proceeding with a renal cell carcinoma treatment option based on thepresence, absence, or level of CD14 cells present within the tumor orperitumoral tissue.

As described herein, the presence of a moderate to marked level of CD14⁺cells within tumor or peritumoral tissue of a renal cell carcinomapatient can indicate that it is unlikely that the renal cell carcinomapatient will respond positively to a type A treatment and that the renalcell carcinoma patient should proceed with a type B treatment. Ingeneral, a moderate level of CD14⁺ cells within tumor tissue orperitumoral tissue is a level where between about 10 and about 30percent of the cells are CD14⁺ cells, while a marked level of CD14⁺cells within tumor tissue or peritumoral tissue is a level where greaterthan about 30 percent of the cells are CD14⁺ cells. Typically, a tumorbiopsy sample is obtained from a cancer patient using standard biopsytechniques. The tumor biopsy sample can include tumor tissue,peritumoral tissue, or both tumor tissue and peritumoral tissue. Onceobtained, the sample can be treated such that the level of CD14⁺ cellspresent can be determined. Standard cell staining and immunofluorescencetechniques can be used to determine the presence or absence of amoderate level or marked level of CD14⁺ cells within tissue. Forexample, a standard microscope can be used in connection withimmunohistochemical staining techniques to determine the presence orabsence of a moderate level or marked level of CD14⁺ cells within tissue(FIGS. 1 and 2). In some cases, CD14 expression within tumor tissue,peritumoral tissue, or both tumor tissue and peritumoral tissue can beassessed using a nucleic acid-based assay (e.g., reversetranscriptase-PCR or nucleic acid microarray assays). For example, CD14expression levels within tumor tissue can be determined using amicroarray having the ability to measure the amount of CD14 mRNA.

The presence of at least a moderate level of CD14⁺ cells within tumor orperitumoral tissue of a renal cell carcinoma patient can indicate thatit is likely that the renal cell carcinoma patient will relapse quicklyand should proceed with an experimental or non-standard treatment. Insome cases, presence of at least a moderate level of CD14⁺ cells withintumor or peritumoral tissue of a renal cell carcinoma patient canindicate that it is unlikely that the renal cell carcinoma patient willrespond positively to a type A treatment and that the renal cellcarcinoma patient should proceed with a type B treatment. Examples oftype A treatments can include, without limitation, surgery alone such asa partial nephrectomy, a simple nephrectomy, or a radical nephrectomy.Examples of type B treatments can include, without limitation, internalor external radiation therapies (e.g., x-ray or radioisotope therapy),chemotherapies, biologic therapies (e.g., immuno, antibody, or high doseIL-2 therapy), cell therapies (e.g., stem cell or vaccine therapy),treatment with Sunitinib (SU11248; an oral, small-molecule,multi-targeted receptor tyrosine kinase inhibitor), treatment withSorafenib (BAY 43-9006, a small molecular inhibitor of several tyrosineprotein kinases), treatment with Bevacizumab (a humanized monoclonalantibody that recognizes and blocks vascular endothelial growth factor),treatment with a rapamycin inhibitor (e.g., Temsirolimus (CCI-779) orEverolimus (RAD001)), and combinations thereof. In some cases, a type Btreatment can be surgery (e.g., a partial nephrectomy, a simplenephrectomy, or a radical nephrectomy) in combination with one or moreof the following therapies: an internal or external radiation therapy(e.g., x-ray or radioisotope therapy), chemotherapy, biologic therapy(e.g., immuno, antibody, or high dose IL-2 therapy), cell therapy (e.g.,stem cell or vaccine therapy), treatment with Sunitinib, treatment withSorafenib, treatment with Bevacizumab, and treatment with a rapamycininhibitor (e.g., Temsirolimus (CCI-779) or Everolimus (RAD001)). In somecases, the absence of at least a moderate level of CD14⁺ cells withintumor or peritumoral tissue of a renal cell carcinoma patient (e.g.,patients with low or absent CD14⁺ staining) can indicate that thepatient should be observed but will likely experience a long period oftumor remission.

In some cases, the methods and materials provided herein can be used totreat glioblastoma multiforme (GBM) cancer. For example, the methods andmaterials provided herein can be used to assess a GBM patient for tumoror peritumoral tissue containing CD14⁺ cells. Once assessed, the patientcan be instructed to proceed with a GBM treatment option based on thepresence, absence, or level of CD14⁺ cells present within the GBM tumoror GBM peritumoral tissue. The presence of a moderate to marked level ofCD14⁺ cells within tumor or peritumoral tissue of a GBM patient canindicate that it is unlikely that the GBM patient will respondpositively to conventional (standard of care) therapy (e.g.,temozolomide chemotherapy after surgery and radiotherapy) and should beincluded in an experimental or alternative therapy (e.g., immune basedtherapies including vaccine or antibody therapy, anti-angiogenesistherapies, cell based therapies, or experimental chemotherapies).

In some cases, the methods and materials provided herein can be used totreat ovarian cancer. For example, the methods and materials providedherein can be used to assess an ovarian cancer patient for tumor orperitumoral tissue containing CD14⁺ cells. Once assessed, the patientcan be instructed to proceed with an ovarian cancer treatment optionbased on the presence, absence, or level of CD14⁺ cells present withinthe ovarian tumor or ovarian peritumoral tissue. The presence of amoderate to marked level of CD14⁺ cells within tumor or peritumoraltissue of an ovarian patient can indicate that it is unlikely that theovarian patient will respond positively to conventional (standard ofcare) therapy and should be included in an experimental or alternativetherapy.

This document also provides methods and materials for identifyingtreatment agents that are capable of reducing cancer aggressiveness(e.g., RCC aggressiveness). For example, patients having moderate tomarked level of CD14⁺ cells within tumor or peritumoral tissue can beadministered a potential treatment agent (e.g., small moleculecompound). After administration of a potential treatment agent for aperiod of time (e.g., once a day to once a week for about two to threemonths), a tumor or peritumoral tissue biopsy can be obtained andassessed for a moderate to marked level of CD14⁺ cells. A reduction inthe level of CD14⁺ cells within the tumor or peritumoral tissue ascompared to the level determined prior to receiving the potentialtreatment agent can indicate that the potential treatment agent is aneffective agent for reducing cancer aggressiveness.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Intratumoral and Peritumoral CD14 Expression AmongPatients with Clear Cell Renal Cell Carcinoma

In a pilot study, ccRCC tumors with high Stage, Size, Grade, andNecrosis (SSIGN) scores were found to have significantly higher levelsof CD14 expression compared with tumors with low SSIGN scores (Table 1).Briefly, CD14 expression was evaluated in 50 clear cell renal cellcarcinoma (ccRCC) cases. The samples were randomly selected from a ccRCCcohort and consisted of 25 cases with low (0-2) and 25 cases with high(7+) SSIGN scores (Frank et al., J. Urology, 168:2395 (2002)).Intratumoral and peritumoral CD14 expression was quantitated as absent,focal, moderate, or marked.

TABLE 1 CD14 expression in 50 ccRCC cases. SSIGN Score Low High CD14Expression N = 25 N (%) N = 25 P-value Intratumoral Absent 2 (8) 1 (4)0.165 Focal 14 (56) 8 (32) Moderate 7 (28) 9 (36) Marked 2 (8) 7 (28)Intratumoral Absent/Focal 16 (64) 9 (36) 0.048 Moderate/Marked 9 (36) 16(64) Peritumoral (N = 46) Absent 2 (8) 0 <0.001 Focal 19 (79) 6 (27)Moderate 3 (13) 10 (46) Marked 0 6 (27) Peritumoral (N = 46)Absent/Focal 21 (87) 6 (27) <0.001 Moderate/Marked 3 (13) 16 (73) Max ofIntratumoral and Peritumoral Absent 1 (4) 0 0.017 Focal 13 (52) 5 (20)Moderate 9 (36) 11 (44) Marked 2 (8) 9 (36) Max of Intratumoral andPeritumoral Absent/Focal 14 (56) 5 (20) 0.009 Moderate/Marked 11 (44) 20(80)

In another study, the associations of tumor CD14 expression withclinical and pathologic features and patient outcome were determinedusing a large cohort of ccRCC patients.

Patient Selection

Using the Mayo Clinic Nephrectomy Registry, 375 patients treated withradical or partial nephrectomy for unilateral, sporadic, non-cysticccRCC were identified.

Clinical and Pathologic Features

The clinical features studied included age, gender, and symptoms atpresentation. Patients with a palpable flank or abdominal mass,discomfort, gross hematuria, acute onset varicocele, or theconstitutional symptoms of rash, sweats, weight loss, fatigue, earlysatiety, or anorexia were considered symptomatic at presentation. Thepathologic features studied included histologic subtype classifiedaccording to the Union Internationale Contre le Cancer, American JointCommittee on Cancer, and Heidelberg guidelines, the 2002 primary tumorclassification, regional lymph node involvement, distant metastases, the2002 TNM stage groupings, tumor size, nuclear grade, coagulative tumornecrosis, sarcomatoid differentiation, and SSIGN score. The SSIGN scoreis a composite scoring system developed specifically for patients withccRCC based on primary tumor classification, regional lymph nodeinvolvement, distant metastases, tumor size, nuclear grade, andcoagulative tumor necrosis. To obtain these features, a pathologistreviewed the microscope slides from all specimens without knowledge ofpatient outcome.

Patient Outcome

Vital status for patients in the Nephrectomy Registry is updated eachyear. If a patient died in the previous year, a death certificate wasordered to determine the cause of death. A visit to the institutionwithin six months of the date of death for metastatic RCC was gooddocumentation that RCC was the cause of death. If the death certificatedoes not support this, the medical history was reviewed by a urologistto determine the cause of death. If a death certificate cannot beobtained, the cause of death was verified with the patient's family orlocal physician.

Immunohistochemical Staining and Quantitation of CD14 Expression

A formalin-fixed, paraffin-embedded block with representative tumortissue was selected for each patient in the cohort. Staining for CD14was conducted as follows. Tissue sections were deparaffinized in xyleneand rehydrated in a graded series of ethanols. Antigen-retrieval wasperformed by heating tissue sections in Target Retrieval solution pH 6(Dako # S1699) to 121° C. using a Digital Decloaking Chamber (BiocareMedical, Walnut Creek, Calif.). Sections were cooled to 90° C. andincubated for an additional five minutes before the Decloaking Chamberwas opened. Sections were washed in running DH₂O for five minutes andincubated for five minutes in Wash Buffer (Dako # S3006) before beingplaced on the Autostainer Plus (Dako) for the following protocol.Sections were blocked for endogenous peroxidase for five minutes usingEndogenous Blocking solution (Dako # S2001), washed twice in washbuffer, incubated five minutes in Background Reducer Sniper (BiocareMedical # BS966L), washed twice in wash buffer, and incubated for 60minutes in purified rabbit anti-human CD14 antibody (Sigma PA #HPA001882) diluted 1:300 with DaVinci Green antibody diluent (BiocareMedical # PD900M). Sections were washed twice in wash buffer, incubated15 minutes in the rabbit probe from the Mach 3 Rabbit HRP-Polymer Kit(Biocare Medical # M3R531L), washed twice in wash buffer, and incubated15 minutes with the rabbit polymer from the Rabbit HRP-Polymer Kit.Sections were washed with wash buffer and visualized by incubatingBetazoid DAB (Biocare Medical # BDB2004L) for eight minutes. Sectionswere washed with DH₂O, counterstained with hematoxylin, dehydrated inethanol, and cleared in xylene. Coverslips were mounted in permanentmounting media. All stained slides were reviewed and quantitated by apathologist without knowledge of patient outcome. Intratumoral and (ifpossible) peritumoral CD14 expression was assessed as absent, focal,moderate, or marked.

Statistical Methods

Associations of CD14 expression with clinical and pathologic featureswere evaluated using chi-square tests. Associations of CD14 expressionwith cancer-specific survival were illustrated using Kaplan-Meiercurves. The magnitudes of the associations of CD14 expression with deathfrom RCC were evaluated using Cox proportional hazards regression modelsand summarized with hazard ratios (HRs) and 95% confidence intervals(CI). The duration of follow-up was calculated from the date ofnephrectomy to the date of death or last follow-up. Patients who diedfrom causes other than RCC were censored at the date of death, whilepatients who were still alive at the time of analysis were censored atthe date of last follow-up. Statistical analyses were performed usingthe SAS software package (SAS Institute; Cary, N.C.). All tests weretwo-sided, and p-values <0.05 were considered statistically significant.

Results

Clinical and Pathologic Features

A summary of clinical and pathologic features for the 375 ccRCC patientsstudied is provided in Table 2. Mean age at surgery was 63 years (median63; range 26-87); mean tumor size was 6.8 cm (median 5.8; range1.0-22.0); and mean SSIGN Score was 3.9 (median 3; range 0-15).

TABLE 2 Clinical and pathologic features for 375 ccRCC patients. FeatureN (%) Age at surgery (years) <65 204 (54.4) ≥65 171 (45.6) Gender Female113 (30.1) Male 262 (69.9) Symptoms 192 (51.2) Constitutional symptoms50 (13.3) 2002 Primary tumor classification pT1a 114 (30.4) pT1b 94(25.1) pT2 71 (18.9) pT3a 40 (10.7) pT3b 50 (13.3) pT3c 3 (0.8) pT4 3(0.8) Regional lymph node involvement pNX and pN0 349 (93.1) pN1 and pN226 (6.9) Distant metastases M0 324 (86.4) M1 51 (13.6) 2002 TNM stagegroupings I 203 (54.1) II 54 (14.4) III 62 (16.5) IV 56 (14.9) Tumorsize (cm) <5 144 (38.4) 5 to <7 73 (19.5) 7 to <10 75 (20.0) ≥10 83(22.1) Nuclear grade 1 24 (6.4) 2 136 (36.3) 3 183 (48.8) 4 32 (8.5)Coagulative tumor necrosis 97 (25.9) Sarcomatoid differentiation 17(4.5) SSIGN score Low (0-2) 175 (46.7) Intermediate (3-6) 111 (29.6)High (≥7) 89 (23.7)CD14 Expression

Intratumoral, peritumoral, and maximal CD14 expression between the twoare summarized in Table 3. A comparison of intratumoral and peritumoralexpression is provided in Table 4. Associations of maximal CD14expression with clinical and pathologic features are summarized in Table5. Since only six tumors did not demonstrate intratumoral or peritumoralCD14 expression, these tumors were combined with those with focalexpression for analysis. CD14 expression, particularly marked CD14expression, was significantly associated with symptoms at presentation,larger tumors of later stage and higher grade, coagulative tumornecrosis, sarcomatoid differentiation, and higher SSIGN scores. Forexample, median SSIGN scores for tumors with absent/focal, moderate, andmarked CD14 expression were 1, 5, and 7, respectively.

TABLE 3 CD14 expression for 375 ccRCC patients. CD14 Expression N (%)Intratumoral Absent 11 (2.9) Focal 224 (59.7) Moderate 100 (26.7) Marked40 (10.7) Peritumoral (N = 373) Absent 9 (2.4) Focal 204 (54.7) Moderate133 (35.7) Marked 27 (7.2) Maximal Intratumoral and Peritumoral Absent 6(1.6) Focal 178 (47.5) Moderate 142 (37.9) Marked 49 (13.1)

TABLE 4 CD14 expression for 375 ccRCC patients. Peritumoral IntratumoralMissing Absent Focal Moderate Marked Absent 0 6 5 0 0 Focal 0 3 170 51 0Moderate 1 0 27 63 9 Marked 1 0 2 19 18

TABLE 5 Associations of CD14 expression with clinical and pathologicfeatures for 375 ccRCC patients. Maximal Intratumoral and PeritumoralCD14 Moderate Absent/Focal N = 142 Marked Feature N = 184 N (%) N = 49P-value Age at surgery (years) <65 109 (59.2) 73 (51.4) 22 (44.9) 0.133≥65 75 (40.8) 69 (48.6) 27 (55.1) Gender Female 64 (34.8) 35 (24.7) 14(28.6) 0.137 Male 120 (65.2) 107 (75.4) 35 (71.4) Symptoms 74 (40.2) 88(62.0) 30 (61.2) <0.001 Constitutional symptoms 13 (7.1) 20 (14.1) 17(34.7) <0.001 2002 Primary tumor classification pT1a and pT1b 138 (75.0)60 (42.3) 10 (20.4) <0.001 pT2 27 (14.7) 30 (21.1) 14 (28.6) pT3a, pT3b,pT3c, and pT4 19 (10.3) 52 (36.6) 25 (51.0) Regional lymph nodeinvolvement pNX and pN0 179 (97.3) 128 (90.1) 42 (85.7) 0.004 pN1 andpN2 5 (2.7) 14 (9.9) 7 (14.3) Distant metastases M0 172 (93.5) 118(83.1) 34 (69.4) <0.001 M1 12 (6.5) 24 (16.9) 15 (30.6) 2002 TNM stagegroupings I 136 (73.9) 58 (40.9) 9 (18.4) <0.001 II 25 (13.6) 22 (15.5)7 (14.3) III 10 (5.4) 34 (23.9) 18 (36.7) IV 13 (7.1) 28 (19.7) 15(30.6) Tumor size (cm) <5 100 (54.4) 37 (26.1) 7 (14.3) <0.001 5 to <735 (19.0) 28 (19.7) 10 (20.4) 7 to <10 31 (16.9) 32 (22.5) 12 (24.5) ≥1018 (9.8) 45 (31.7) 20 (40.8) Nuclear grade 1 17 (9.2) 7 (4.9) 0 <0.001 299 (53.8) 34 (23.9) 3 (6.1) 3 66 (35.9) 87 (61.3) 30 (61.2) 4 2 (1.1) 14(9.9) 16 (32.7) Coagulative tumor necrosis 12 (6.5) 53 (37.3) 32 (65.3)<0.001 Sarcomatoid differentiation 1 (0.5) 7 (4.9) 9 (18.4) <0.001 SSIGNscore Low (0-2) 128 (69.6) 44 (31.0) 3 (6.1) <0.001 Intermediate (3-6)42 (22.8) 53 (37.3) 16 (32.7) High (≥7) 14 (7.6) 45 (31.7) 30 (61.2)Patient Outcome

At last follow-up, 159 patients had died, including 100 who died fromRCC at a mean of 2.3 years following surgery (median 1.7; range 0-9).Among the 216 patients who were still alive at last follow-up, the meanduration of follow-up was 7.1 years (median 7.3; range 0-10); only 5(2.3%) patients had fewer than 2 years of follow-up. Estimatedcancer-specific survival rates (95% CI, number still at risk) at 1, 3,5, and 7 years following surgery were 91.0% (88.1-94.0; 328), 79.0%(74.9-83.4; 268), 75.3% (70.9-80.0; 232), and 71.9% (67.3-77.0; 136),respectively.

The association of maximal intratumoral and peritumoral CD14 expressionwith cancer-specific survival was determined (FIG. 3). The magnitudes ofthe associations of CD14 expression with death from RCC are summarizedin Table 6. Univariately, patients whose tumors contained moderate CD14expression were nearly five times more likely to die from RCC comparedwith patients whose tumors contained absent/focal CD14 expression (HR4.79; p<0.001). Patients whose tumors contained marked CD14 expressionwere over 11 times more likely to die from RCC compared with patientswhose tumors contained absent/focal CD14 expression (HR 11.51; p<0.001).These associations were also evaluated in a multivariable settingadjusting for the SSIGN score. After accounting for the associationbetween SSIGN score and death from RCC, the HRs for the associations ofmoderate and marked CD14 expression with death from RCC were 2.87(p<0.001) and 3.03 (p<0.001), respectively. After further adjustment forage at surgery, gender, symptoms at presentation, sarcomatoiddifferentiation, and the SSIGN score, the HRs for the associations ofmoderate and marked CD14 expression with death from RCC were 2.53 (95%CI 1.45-4.42; p=0.001) and 2.69 (1.44-5.01; p=0.002), respectively.

TABLE 6 Univariate and multivariable associations of CD14 expressionwith death from RCC for 375 patients with ccRCC. CD 14 Expression HR(95% CI) P-value Univariate Maximal Intratumoral and PeritumoralAbsent/Focal 1.0 (reference) Moderate 4.79 (2.76-8.32) <0.001 Marked11.51 (6.39-20.73) <0.001 Adjusted for SSIGN Score Maximal Intratumoraland Peritumoral Absent/Focal 1.0 (reference) Moderate 2.87 (1.65 -5.00)<0.001 Marked 3.03 (1.65-5.58) <0.001

Example 2—Treating Renal Cell Carcinoma Patients

A newly diagnosed RCC patient who is currently not being treated and whohas a high level of CD14⁺ cells within tumor or peritumoral tissue istreated with standard of care (e.g., surgery), but is subjected toincreased surveillance as this patient is highly likely to relapseand/or experience metastasis.

A newly diagnosed RCC patient who is currently not being treated and whohas no CD14⁺ cells within tumor or peritumoral tissue is treated bysurgery if indicated and observation.

An RCC patient who is currently being treated with a standard cancertreatment and who has a high level of CD14⁺ cells within tumor orperitumoral tissue is included in a clinical trial or other aggressivetherapy without waiting for relapse.

An RCC patient who is currently being treated with a standard cancertreatment and who has no CD14⁺ cells within tumor or peritumoral tissueis under a routine schedule of observation without additional treatment.

Example 3—Correlation Between Intratumoral CD14⁺ Cell Levels andCD14/DR⁻ Peripheral Blood Levels

A group of 17 patients had tumor samples that were assessed for thelevel of CD14+ cells and had peripheral blood samples that were assessedfor CD14 and HLA-DR staining. The tumor samples were classified ashaving a high intensity of CD14⁺ staining (high), a medium intensity ofCD14⁺ staining (medium), or a low/no intensity of CD14⁺ staining(low/none). The peripheral blood samples were assessed to determine themean fluorescence intensity (MFI) of HLA-DR⁺ staining for peripheralblood cells that are CD14⁺.

As the level of CD14⁺ staining increases in intratumoral tissue, moreCD14⁺ cells in the periphery lose HLA-DR expression (FIG. 4). Theseresults demonstrate that there is an association between the level ofintratumoral CD14⁺ cells and the level of peripheral CD14+/DR⁻ cells.These results also demonstrate that the level of CD14+/DR⁻ cells can beused to measure the intratumoral level of CD14⁺ cells, and therefore bean accurate surrogate for the tumor staining.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a human having an ovariantumor, the method comprising administering an immune-based therapy, ananti-angiogenesis therapy, or a cell-based therapy to a human who wasdetermined to have an ovarian tumor wherein greater than 30 percent ofthe cells of the tumor or peritumoral tissue are CD14⁺ cells.